BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 234, 269274 (1997)

ARTICLE NO. RC976622

ElectrolyzedReduced Water Scavenges Active Oxygen

Species and Protects DNA from Oxidative Damage
Sanetaka Shirahata,1 Shigeru Kabayama, Mariko Nakano, Takumi Miura,
Kenichi Kusumoto, Miho Gotoh, Hidemitsu Hayashi,* Kazumichi Otsubo,**
Shinkatsu Morisawa,** and Yoshinori Katakura
Institute of Cellular Regulation Technology, Graduate School of Genetic Resources Technology, Kyushu University, 6-10-1
Hakozaki, Higashi-ku, Fukuoka 812-81, Japan; *Water Institute, Nisshin Building 9F, 2-5-10 Shinjuku, Tokyo 160,
Japan; and **Nihon Trim Co. Ltd., Meiji Seimei Jusou Building 6F, 1-2-13 Shinkitano, Yodogawa-ku, Osaka 532, Japan

Received March 21, 1997

ascorbic acid, could directly scavenge H2O2 . Reduced

Active oxygen species or free radicals are considered
to cause extensive oxidative damage to biological mac-
romolecules, which brings about a variety of diseases
as well as aging. The ideal scavenger for active oxygen
should be active hydrogen. Active hydrogen can be not only O0
produced in reduced water near the cathode during
electrolysis of water. Reduced water exhibits high pH,
low dissolved oxygen (DO), extremely high dissolved
molecular hydrogen (DH), and extremely negative re-
dox potential (RP) values. Strongly electrolyzedre-
duced water, as well as ascorbic acid, (/)-catechin and
tannic acid, completely scavenged O0 2 produced by the
hypoxanthine-xanthine oxidase (HX-XOD) system in
sodium phosphate buffer (pH 7.0). The superoxide dis-
mutase (SOD)-like activity of reduced water is stable
at 47C for over a month and was not lost even after
neutralization, repeated freezing and melting, defla-
tion with sonication, vigorous mixing, boiling, re-
peated filtration, or closed autoclaving, but was lost
by opened autoclaving or by closed autoclaving in the
presence of tungsten trioxide which efficiently ad-
sorbs active atomic hydrogen. Water bubbled with hy-
drogen gas exhibited low DO, extremely high DH and
extremely low RP values, as does reduced water, but
it has no SOD-like activity. These results suggest that
the SOD-like activity of reduced water is not due to the
dissolved molecular hydrogen but due to the dissolved
atomic hydrogen (active hydrogen). Although SOD ac-
cumulated H2O2 when added to the HX-XOD system,
reduced water decreased the amount of H2O2 produced
by XOD. Reduced water, as well as catalase and
1
To whom correspondence should be addressed. Fax: 81-92-642-
3052. E-mail: sirahata@grt.kyushu-u.ac.jp.
Abbreviations: AET, 2-(aminoethyl)isothiuronium; AsA, ascorbic
acid; CL, chemiluminescence; CLA, Cypridina luciferin analog; DO,
dissolved oxygen; DH, dissolved hydrogen, EC, electrical conduc-
tance; HX, hypoxanthine; RP, redox potential; SOD, superoxide dis-
mutase; XOD, xanthine oxidase.
water suppresses single-strand breakage of DNA by
active oxygen species produced by the Cu(II)-cata-
lyzed oxidation of ascorbic acid in a dose-dependent
manner, suggesting that reduced water can scavenge
2 and H2O2 , but also 1O2 and OH. q 1997
Academic Press
Active oxygen species or free radicals, such as singlet
oxygen (1O2), superoxide anion radical (O02 ), hydrogen
peroxide (H2O2) and hydroxyl radical (OH) are consid-
ered to cause extensive oxidative damage to biological
macromolecules (DNA, membrane polyunsaturated
fatty acid chains, enzymes and so on), which bring
about a variety of diseases, as well as aging (1, 2). We
believe that the ideal countermeasure against active
oxygen is active hydrogen. Electrolysis of water pro-
duces reduced and oxidized water near the cathode and
anode, respectively. Reduced water exhibits high pH,
low dissolved oxygen (DO), high dissolved hydrogen
(DH) and significant negative redox potential (RP) val-
ues. Soft water in Japan made it possible to develop
domestic devices to reform water by electrolysis about
half a century ago.
So far, the characteristics of neither reduced water
nor oxidized water have been well clarified. Based upon
the interesting clinical improvement of a variety of dis-
eases by intake of reduced water since 1985, Hayashi
proposed the hypothesis Water Regulating Theory (3).
Here, based on his theory we first demonstrate that
reduced water scavenges active oxygen species and pro-
tects DNA from damage by oxygen radicals.
MATERIALS AND METHODS
Electrolysis of water. Ultrapure water produced by an ultrapure
system (ULTRAPUR LV-10T, TORAY, Tokyo) was added 0.1 g/l NaCl

269 0006-291X/97 $25.00

Copyright q 1997 by Academic Press
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to elevate electrical conductance (EC) to about 20 ms/m. The water

was then electrolyzed with various voltages by an electrolyzing de-
vice (Type TI-7000S and TI-7000SL, Nihon Trim Co., Osaka)
equipped with a platinum-coated titanium electrode to produce re-
duced water which exhibited various RP. RP, EC, DO and DH were
measured using a RP meter (type, HM-14P), a EC meter (CM-14P),
a DO meter (DO-14P) and a DH meter (DHDI-1) from Toa Electronics
Ltd. (Tokyo) at 257C. pH was measured using a pH meter (Beckman,
Type pHI32) at 257C.
Assay of the SOD-like activity of reduced water, ascorbic acid (AsA),
(/)-catechin, and tannic acid. The reaction mixture (1 ml) for mea-
suring chemiluminiescence (CL) intensity specific to O20 contained
500 mM hypoxanthine (HX), 200 mM EDTA, 40 mM sodium phos-
phate buffer (pH 7.0), 0.6 ml of the reduced water or NaOH solution
of the same pH as reduced water, 2.5 mM 2-methyl-6-phenyl-3,7-
dihydroimidazo[1,2-a] pyrazin-3-one (Cypridina luciferin analog
(CLA), Tokyo Kasei Industrial Co., Tokyo) and 0.5 U/l of xanthine
oxidase (XOD) (Wako Purechemical Industries, Tokyo). The CL in-
tensity of the reaction mixture (0.85 ml) except for XOD solution was
measured in a glass-tube in a CL reader (Aloka, type BLR-301) at
26 7C. At 18 seconds time point, 0.15 ml of XOD solution was injected
into the tube and the CL intensity was continuously measured for
120 sec. The change of the CL intensity in the presence of reduced
water, SOD derived from bovine erythrocytes (Sigma), AsA (Wako),
(/)-catechin (Wako) or tannic acid (Wako) was measured with the
HX-XOD system to evaluate the scavenging activity of O20 . The
strongly reduced water (RP, 0820 mV; pH 11.0) was diluted with a
NaOH solution (pH 11.0) to assure the same final pH of the reaction
mixture (pH 7.3) among the test samples. The extinguishing rate of
O20 (%) was calculated by dividing the total CL intensity of the test
sample by that of the control.
Analysis of catalase-like activity of reduced water and AsA. The
reaction mixture (100 ml) of the HX-XOD system described above in
the presence or absence of SOD or reduced water was put into a 96-
well microplate and incubated at 37 7C. Time course of the change
in the H2O2 concentration was measured by the addition of 200 ml
of substrate containing peroxidase (0.3 mg/ml 2,2*-azino-di-(3-ethyl
benzthiazolin sulfonic acid), 0.1 M citrate buffer, pH 4.0, streptavi-
dine-horseradish peroxidase conjugate (Amersham) diluted to 1000
times). Absorbency of the reaction product was measured at 405 nm
by an ELISA reader. In order to examine the catalase-like activity,
the H2O2 solution was directly incubated with control NaOH solution
(pH 11.0), reduced water (pH 11.0), AsA or catalase (Wako) in 40
mM sodium phosphate buffer (pH 7.0) at 37 7C and the H2O2 concen- FIG. 1. Relationships of RP with pH (A), DO (B) and DH (C) in
trations were determined. electrolyzed-reduced water. Ultrapure water was electrolyzed by an
Analysis of single-strand breakage of DNA caused by active oxygen electrolyzing device and RP, pH, DO and DH values were immedi-
species produced by the Cu(II)-catalyzed oxidation of AsA. Time- ately measured. The data of water before electrolysis was shown by
course of the single-strand DNA breaking reaction caused by Cu(II)- dark squares. Arrows show the reduced water which exhibited the
catalyzed oxidation of AsA was examined by using super-coil plas- strong SOD-like activity.
mid DNA. One mg of pBluescript II plasmid DNA (Stratagene, La
Jolla, CA) was incubated with the mixture of 25 mM AsA and 25
mM CuSO4 in 20 ml of 20 mM sodium phosphate buffer (pH 7.5)
containing 17 ml of reduced water, NaOH solution of the same pH the cathode and oxidation at the anode. Dissociation of
as the reduced water, SOD, catalase, 2-(Aminoethyl)isothiuronium H2O produces H/ and OH0 ions. At the cathode, H/
(AET)(Wako), KI (Wako), or NaN3 (Wako) at 377C for various peri-
ions gain electrons to change into active atomic hydro-
ods of time. The reaction was stopped by adding 4 ml of 15 mM
EDTA (pH 7.0). Super-coil DNA, open-circular DNA and linear DNA gen (H). Active atomic hydrogen exhibits high reducing
were separated by electrophoresis with 1% agarose gel and any potential. It is then changed to hydrogen molecules (H2)
changes of the amount of super-coil DNA were determined from the which are chemically inert at room temperature. At
photo images by a NIHimage computer software. the anode, OH0 ions lose electrons to form OH, which
results in the production of O2 and H2O. Cathodic alka-
RESULTS AND DISCUSSION line water (reduced water) is abundant in DH, whereas
anodic acidic water (oxidized water) is abundant in DO.
Characteristics of electrolyzedreduced water. The The relationships of RP with pH, DO, and DH in re-
principle of electrolysis was founded by Michael Fara- duced water were shown in FIG. 1. Marked changes in
day (1791-1867). In this process, reduction occurs at these values occur in water after electrolysis. It should
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Vol. 234, No. 1, 1997 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
FIG. 2. Analysis of the SOD-like activity of reduced water.
(A) Effects of reduced water, SOD, H2-water and N2-water on the
accumulation of O20 generated by the HX-XOD system. The CL
intensity was determined in phosphate buffer (pH 7.0) in the pres-
ence of reduced water (RP 0820 mV, pH 11.0; IC50SO(18%)), a
NaOH solution of the same pH as reduced water, SOD, H2-water,
or N2-water. h, control NaOH solution; j, reduced water; m, SOD
(0.13 U/ml); s, SOD (66.7 U/ml); l, H2-water; n, N2-water. (B)
Stability of the SOD-like activity of reduced water. h, control
NaOH solution. l, reduced water and reduced water treated with
repeated freezing and melting, deflation with sonication, vigorous
mixing for 10 minutes, boiling for 10 minutes, repeated filtration
with 0.22mm filter, or closed autoclaving at 1217C for 20 minutes.
m, reduced water treated with opened autoclaving at 1217C for 20
minutes. n, reduced water treated with closed autoclaving in the
presence of tungsten trioxide (0.5 g/70ml reduced water) at 1217C
for 20 minutes. (C) The SOD-like activity of diluted reduced water.
Amount of reduced water (%): h, 60%; /, 30%; *, 26%; m, 23%; h,
21%; L, 20%; l, 19%; ,, 15%; s, 7.5%; , 0% (control). (D) Inhibi-
tion of the accumulation of O20 by variously diluted reduced water.
All experiments were triplicated and the average values were
shown in the figures. The SD errors were within 5%.
be noticed that the DH value is higher in reduced water
than in the original water by two orders of magnitude.
The SOD-like activity of reduced water. XOD oxi-
dizes HX to xanthine, coupling to generation of O0 2
from O2 . CLA specifically reacts with O0 1
2 and O2 and
emits a CL (4). As shown in FIG. 2A, reduced water
completely inhibited the CL, demonstrating the SOD-
like activity of reduced water. Since the CL was com-
pletely inhibited by SOD, the CL was specific to O0
2 .
Bubbling of hydrogen gas into a NaOH solution (pH
10.5) for 5 minutes resulted in remarkable changes of
RP from /116 mV to 0842 mV; DO from 7.66 mg/l to
1.98 mg/l; DH from 0.0002 mg/l to 0.938 mg/l; but no
change of pH. Bubbling of nitrogen gas into a NaOH
solution (pH 10.5) resulted in changes of RP from /116
mV to /83 mV; DO from 7.66 mg/l to 1.82 mg/l; DH
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from 0.0002 mg/l to 0.0001 mg/l; but no change of pH.
As shown in FIG. 2A, water bubbled with hydrogen gas
(H2-water) and nitrogen gas (N2-water) showed de-
creased CL intensity by about 20% as compared to that
of the control in the steady state, suggesting that the
decreased CL intensity in N2-water and H2-water was
due to low DO and H2-water had no SOD-like activity.
The SOD-like activity of reduced water is retained dur-
ing storage, although the RP and DH values exhibit
decay. These results clearly indicate that dissolved mo-
lecular hydrogen gas in reduced water was responsible
for the negative RP value, but not for the SOD-like
activity.
Stability of the SOD-like activity of reduced water.
The SOD-like activity of reduced water was very stable
in a closed glass bottle at 47C for over a month. The
activity was not lost even after neutralization, repeated
freezing and melting, deflation with sonication for 10
minutes, repeated filtration with a 0.22 mm filter, boil-
ing for 10 minutes, or autoclaving in a closed glass
bottle at 1217C for 20 minutes (FIG. 2B). However, 90%
the SOD-like activity was lost by autoclaving in an
opened glass bottle, suggesting that the active sub-
stance in the reduced water is volatile. Atomic hydro-
gen is volatile and can reduce metallic oxide such as
tungsten trioxide, though molecular hydrogen cannot
easily do this (5). A sensitive detection method of
atomic hydrogen is based on the color change of tung-
sten trioxide by reduction (6). The closed autoclaving
of reduced water with tungsten trioxide at 1217C for
20 minutes resulted in the loss of 52% of the SOD-like
activity (FIG. 2B). These results strongly suggest that
the substance responsible for the SOD-like activity of
reduced water is active atomic hydrogen. Active hydro-
gen in gas phase is rather stable taking into consider-
ation of the collision rate (7). Since two atoms of hydro-
gen in the ground state have larger energy than molec-
ular hydrogen in any stable state, the extra energy is
required to be eliminated by a third substance to pro-
duce a hydrogen molecule from two atoms of hydrogen
by collision (8). Atomic hydrogen can stably exist
avoiding the attack by oxygen in solution and crystals
at room temperature for a period longer than a year
(9). Water vapor is known to prevent the recombination
of atomic hydrogen on a tungsten surface (8). The char-
acteristics in aqueous solution of active atomic hydro-
gen produced by electronic discharge is under investi-
gation.
When XOD solution was injected into the reaction
mixture containing the diluted reduced water, strong
CL was emitted immediately after injection (FIG. 2C),
but the intensity of CL rapidly dropped below the con-
trol value. The initial strong transient emission of CL
in diluted reduced water could be inhibited by SOD,
indicating that a large amount of O0 2 was transiently
generated just after the addition of XOD. AsA, (/)-
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catechin and tannic acid, as well as SOD, did not show

such a strong initial transient emission of CL (data not
shown). Injection of the solvent without enzyme did not
result in an emission of CL. More detailed experiments
will be needed to clarify the mechanism of this phenom-
enon. The inhibitory effect of reduced water on the ac-
cumulation of O02 was increased in a dose-dependent
manner as shown in FIG. 2D, suggesting the stoichio-
metric action of active substance in reduced water.
Definition of the SOD-like activity of reduced water.
Since this paper first reports the O0
2 scavenging activ-
ity of electrolyzed-reduced water, the standardization
of this activity is needed to compare the reducing po-
tency of the reduced water prepared each time. In order
to standardize the reducing potency of the reduced wa-
ter, we defined a IC50SO unit as a reducing potency of
which reduced water can scavenge 50% of O0 2 gener-
ated by the HX-XOD system under the conditions de-
scribed in the MATERIALS AND METHODS section
and a IC50SO (%) as the concentration (%) of reduced
water in which the 50% of O0 2 generated by the HX-
XOD system is scavenged. The IC50SO values of SOD,
AsA, (/)-catechin, and tannic acid were 0.05 U/ml, 3
mM (0.53 mg/ml), 130 mM (38 mg/ml), and 33 mg/ml in
the HX-XOD system used here.
Catalase-like activity of reduced water. Although
reduced water could scavenge O0 2 , there was a possi-
bility that reduced water inhibited the enzyme activity
of XOD or inhibited the reaction between O0 2 and the
luciferin analog reagent. To eliminate this possibility,
the production of H2O2 was determined in the HX-XOD
system. XOD can produce not only O0 2 but also H2O2
in this HX-XOD system. As shown in FIG. 3A, XOD
produced H2O2 even in the presence of reduced water,
FIG. 3. The SOD-like and catalase-like activity of reduced water
demonstrating no inhibition of the enzyme activity by and AsA. (A) Change of the amount of H2O2 in the HX-XOD system
reduced water. As expected, the addition of SOD to the in the presence of SOD or reduced water. SOD (70 U/ml) or reduced
HX-XOD system resulted in the accumulation of H2O2 , water (12 IC50SO units) were incubated in the HX-XOD system at
indicating that O0
2 produced by XOD was changed to
37 7C. h, control NaOH; L, reduced water; s, SOD. (B) Degradation
H2O2 by SOD. Reduced water first accumulated H2O2 of H2O2 by reduced water, AsA and catalase. Reduced water (12
IC50SO units), AsA (100 mM) or catalase (20 U/ml) was incubated
and then gradually lowered the concentration of H2O2 , with H2O2 (70 mM) in 20 mM sodium phosphate buffer (pH 7.5) at
suggesting that reduced water exhibits not only SOD- 37 7C. h, control; L, reduced water; n, AsA; s, catalase. All experi-
like activity but also catalase-like activity. The fact ments were triplicated and the average values were shown. The stan-
that reduced water stimulated the accumulation of dard errors were shown by vertical bars. In (B) most of the SD error
bars are embedded in the symbols.
H2O2 in the HX-XOD system in the first 5 minutes
indicated that the decreased CL intensity in the pres-
ence of reduced water is not due to the inhibition of
the reaction between O0 2 and luciferin analog by re- catalyzed oxidation of AsA is known to produce O0 2
duced water, but due to the conversion of O0
2 into H2O2 and H2O2 which react to produce OH (12). In order to
by reduced water. To demonstrate the catalase-like ac- demonstrate that reduced water can scavenge not only
tivity of reduced water, H2O2 was directly incubated O0
2 and H2O2 but also other active oxygen species, we
with reduced water. As shown in FIG. 3B, reduced wa- examined the effect of reduced water on DNA breakage
ter scavenged H2O2 as well as AsA and catalase. caused by the mixture of AsA and Cu(II). Super-coil
Suppressive effect of reduced water on the single- plasmid DNA (Form I) changes to open-circular DNA
strand breakage of DNA caused by the Cu(II)-catalyzed (Form II) by a single-strand breakage. Open-circular
oxidation of AsA. The DNA strand breakage is caused DNA changes to linear DNA (Form III) by a double-
by the mixture of AsA and Cu(II) (10, 11). The Cu(II)- strand DNA breakage. When plasmid DNA was incu-
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bated with the mixture of AsA and Cu(II), the amount TABLE 1
of super-coil DNA gradually decreased (FIG. 4). How- Effect of Reduced Water, SOD, Catalase, and Various
ever, reduced water significantly inhibited the single- Radical Scavengers on Single-Strand Breakage of DNA by
strand breakage by the mixture of AsA and Cu(II). As the Cu(II)-Catalyzed Oxidation of AsA
shown in Table 1, reduced water inhibited the DNA
Addition Concn Specificity Inhibin, %
breaking reaction in a dose-dependent manner. Cata-
lase also inhibited the breaking reaction but SOD did Reduced water 3.7 IC50SO units 19
7.5 IC50SO units 38
15 IC50SO units 49
SOD 150 U/ml O20 2
Catalase 0.8 U/ml H2O2 6
4 U/ml H2O2 36
20 U/ml H2O2 90
AET a 4 1 1005 M gereral 44

KI 1 1 1002 M OH 18

5 1 1002 M OH 53
1
NaN3 1 1 1002 M O2 14
1
5 1 1002 M O2 43
a
AET 2-(aminoethyl)isothiuroium.

not, indicating that H2O2 participated in the breaking

FIG. 4. Inhibitory effect of reduced water on single-strand break-
age of DNA caused by oxygen radicals produced by the Cu(II)-cata-
lyzed oxidation of AsA. (A) Electrophoresis of plasmid DNA treated
with the mixture of AsA and Cu(II) in the presence of 4.1 IC50SO
units of reduced water (RP, 0659 mV; pH 10.5; IC50SO(18%)) or
NaOH solution (pH 10.5). SC, super-coil DNA. OC, open-circular
DNA. Lane 1, marker (l DNA-HindIII); lane 2, control DNA (2
hours); lane 3, DNA / Cu(II) (2 hours); lane 4, DNA / AsA (2 hours).
Lanes 5, 7, 9, 11 and 13 show the DNA breaking reaction by the
mixture of AsA and Cu(II) in the presence of NaOH solution for 0,
15, 30, 60 and 120 minutes, respectively. Lanes 6, 8, 10, 12 and 14
show the DNA breaking reaction by the mixture of AsA and Cu(II)
in the presence of reduced water for 0, 15, 30, 60 and 120 minutes,
respectively. (B) Decrease of the relative amount (%) of super-coil
DNA during the incubation of plasmid DNA with the mixture of AsA
and Cu(II) in the presence of reduced water (s) or NaOH solution
(l). Average values of two independent experiments (n2, each) were
shown. The vertical bars show the SD errors.
reaction, but O0
2 did not. Both radical scavengers spe-
cific to OH or 1O2 inhibited the breaking reaction.
These results indicated that reduced water can prevent
DNA damage caused by active oxygen species such as
H2O2 , OH, and 1O2 produced by the Cu(II)-catalyzed
oxidation of AsA. Significant synergistic effect between
reduced water and the radical scavengers used here
was not observed (data not shown). It is noteworthy
that reduced water can prevent DNA damage even in
the presence of metallic ions which catalyze the autoox-
idation of most established antioxidants and reverse
their protective effect against active oxygen species.
However, the water must have a higher reducing abil-
ity for this purpose than scavenging O0 2 .
Although aerobic organisms have evolved by acquisi-
tion of the ability to utilize oxygen, oxygen is princi-
pally a toxic substance. Recently biological activation
of hydrogen by hydrogenases was reported (13). Hydro-
genases, which are among in the oldest enzymes (3.8
billion years old), can reversibly split molecular hydro-
gen to produce active atomic hydrogen. Active atomic
hydrogen may have participated in the redox regula-
tion of cellular functions. Water can permeate every-
where in the body and penetrates every membrane in-
cluding the blood-brain barrier. To neutralize the toxic
action of active oxygen species, electrolyzed-reduced
water may be an ideal and very powerful antioxidant.
Further intensive investigation on the effect of reduced
water on cell biology, immunology and oncology should
be promoted.
ACKNOWLEDGMENTS
We are grateful to Dr. David Barnes, Oregon State University,
and Mr. Mark Selby for their critical reading of the manuscript.

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